Abstract Stroke is the nation's third leading cause of death and the most common cause of permanent disability in adults. Thus far, the only drug that has been used successfully to treat acute stroke in the clinic is the clot- dissolving drug tissue plasminogen activator (tPA). However, tPA must be administered within 3 hours of the onset of an ischemic stroke, making it a viable treatment for less than 6% of patients. Thus, strategies providing for a wider inclusion of patient populations are needed for the treatment of stroke. Erythropoietin (EPO) has recently emerged as a promising candidate both for neuroprotection and neurorecovery in ischemic stroke. EPO has robust neuroprotective effects in both in vitro and in vivo models of ischemic injury. A recent clinical trial demonstrated the beneficial effects of administering EPO in patients with acute ischemic stroke. Thus, a neuroprotective approach using EPO in stroke, especially in treatment of patients who are not suitable for tPA treatment, represents a potentially exciting clinical application. Excitingly, our recent study indicated that EPO administration initiated at 48 hours after ischemia did not reduce brain tissue loss, but stimulated neurogenesis and oligodendrogenesis, attenuated white matter injury, and significantly improved neurological recovery after ischemia, suggesting that EPO is a promising therapeutic reagent for the neurological functional recovery of poststroke patients. However, large doses and multiple administration of EPO are required for the treatment of stroke, especially for the delayed treatment. Such a regimen of EPO administration may potentially result in multiple high risk factors for stroke patients, such as increases in hematocrit and quantity of platelets, increasing the likelihood of secondary-infarction. Accordingly, alternate strategies to reduce erythropoietic activity and other potential side effects of EPO will greatly improve its clinical applications for the treatment of stroke patients. We have successfully generated a novel mutant EPO (MEPO) containing a single amino acid mutation which completely lacks erythropoietic activity. Importantly, MEPO has neuroprotective effects and retains the ability to stimulate the neurogenesis with similar efficacies as wild-type EPO. Therefore, the objective of this proposal is to test the neurorecovery effect of MEPO lacking erythropoietic activity after a delayed administration of MEPO in the clinically relevant middle cerebral artery occlusion (MCAO) model. The following specific aims are proposed: Aim 1: To test the hypothesis that delayed administration of MEPO lacking erythropoietic activity improves neurological outcomes after ischemic injury. Focal cerebral ischemia will be induced by MCAO for 60 min. EPO and MEPO will be injected intraperitoneally into mice in a delayed manner. These studies will determine: 1) whether delayed injection of MEPO results in improved neurological outcomes after ischemia, and 2) whether delayed injection of MEPO reduces brain tissue loss. Aim 2: To test the hypothesis that MEPO mediates the neurorecovery effect via enhancing neurovescular remodeling and attenuating white matter injury. We will test: 1) whether delayed injection of MEPO enhances angiogenesis and neurogenesis; 2) whether delayed injection of MEPO attenuates white matter injury and 3) whether delayed administration of MEPO inhibits cell death after ischemia. Aim 3: To test the hypothesis that neurogenesis/oligodendrogenesis effects of MEPO is mediated by EPO/CR heterocomplex via PI3K/Akt and MAPK/Erk1/2 signaling pathways. We will test whether MEPO mediates neurogenesis/oligodendrogenesis via binding to EPO/CR heterocomplex and activating PI3K/Akt and MAPK/Erk1/2 pathways in an in vitro model of neurogenesis in neurosphere cultures as well as in the murine MCAO model.